Modulating apoptosis

ABSTRACT

The use and screening of modulators of apoptosis is disclosed. The modulators may be, for example, modulator of NF-κB activity. The modulators may be used, for example, in the treatment of NF-κB-mediated diseases, conditions, and injuries.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2004/040579, filed Dec. 2, 2004, International PatentApplication No. PCT/US2004/040749, filed Dec. 2, 2004, InternationalPatent Application No. PCT/US2004/040750, filed Dec. 2, 2004, andInternational Patent Application No. PCT/US2004/040753, filed Dec. 2,2004, each of which claims the benefit of U.S. Provisional ApplicationNo. 60/526,460, filed Dec. 2, 2003, U.S. Provisional Application No.60/526,461, filed Dec. 2, 2003, U.S. Provisional Application No.60/526,496, filed Dec. 2, 2003, and U.S. Provisional Application No.60/526,666, filed Dec. 2, 2003, the contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

This invention relates to the modulation of apoptosis.

BACKGROUND OF THE INVENTION

The progression from normal cells to tumor cells involves a loss ofnegative mechanisms of growth regulation, including resistance to growthinhibitory stimuli and a lack of dependence on growth factors andhormones. Traditional cancer treatments that are based on radiation orcytotoxic drugs rely on the differences in growth control of normal andmalignant cells. Traditional cancer treatments subject cells to severegenotoxic stress. Under these conditions, the majority of normal cellsbecome arrested and therefore saved, while tumor cells continues todivide and die.

However, the nature of conventional cancer treatment strategy is suchthat normal rapidly dividing or apoptosis-prone tissues are at risk.Damage to these normal rapidly dividing cells causes the well-known sideeffects of cancer treatment (sensitive tissues: hematopoiesis, smallintestine, hair follicles). The natural sensitivity of such tissues iscomplicated by the fact that cancer cells frequently acquire defects insuicidal (apoptotic) machinery and those therapeutic procedures thatcause death in normal sensitive tissues may not be that damaging tocancer cells. Conventional attempts to minimize the side effects ofcancer therapies are based on (a) making tumor cells more susceptible totreatment, (b) making cancer therapies more specific for tumor cells, or(c) promoting regeneration of normal tissue after treatment (e.g.,erythropoietin, GM-CSF, and KGF).

There continues to be a need for therapeutic agents to mitigate the sideeffects associated with chemotherapy and radiation therapy in thetreatment of cancer. This invention fulfills these needs and providesother related advantages.

SUMMARY OF THE INVENTION

A method of protecting a mammal from a condition that triggers apoptosisis provided. The mammal may be administered a composition comprising anagent that induces NF-κB. The agent may be flagellin or TGFβ, which maybe latent TGFβ.

The condition may be radiation exposure. The composition may beadministered in combination with a radioprotectant, which may be anantioxidant or a cytokine. The antioxidant may be amifostine or vitaminE. The cytokine may be a stem cell factor.

The condition may also be a constitutively active NF-κB cancer. Thecondition may also be a cancer treatment, which may be chemotherapy orradiation therapy. The composition may be administered prior to,together with, or after the cancer treatment.

The condition may also be cell aging, radiation, wounding, poisoning,infection or temperature shock.

Also provided is a method of screening for a modulator of apoptosis. Asuspected modulator may be added to a cell-based apoptosis system. Acontrol may also be added to the cell-based apoptosis system. The levelof apoptosis of the suspected modulator and the control may be comparedto identify a modulator of apoptosis. The suspected modulator may bederived from a mammalian parasite. The modulator of apoptosis may be amodulator of NF-kB, TGFβ, or p53. The cell-based apoptosis system may bea NF-kB-, TGFβ-, or p53-activated expression system. The level ofapoptosis may be the level of NF-kB-, TGFβ-, or p53-activatedexpression. The parasite species may include, but is not limited to,Salmonella, Mycoplasma, or Chlamydia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that Salmonella infection leads to NF-κB nuclearlocalization even in non-infected cells. HT29 cells were grown on glasscoverslips and either mock-infected, left untreated, infected withSalmonella typhimurium, or treated with TNFα (10 ng/ml). Panel A: HT29cells were mock-infected or infected at an MOI of 100 with Salmonellatyphimurium strain SJW1103G which expresses GFP from the ssaH promoterthat is only active inside infected host cells. Cells were photographedusing bright field microscopy (BF), and immunoflourescence to detect GFPor DAPI staining as indicated. Images were merged (overlay) to revealcells that were infected. Panel B: HT29 cells were left untreated,infected with Salmonella typhimurium strain 1103 or treated with TNFα.NF-κB p65(RelA) localization under various conditions as indicated wasmonitored by indirect immunofluorescence. Cells were visualized bybright field microscopy (BF), cell nuclei were stained with DAPI andp65(RelA) was visualized with FITC. DAPI staining was falsely coloredred to make visualization of the merge (overlay) easier to distinguish.

FIG. 2 demonstrates that a protein factor in Salmonella culture brothleads to NF-κB activation. Panel A: Salmonella dublin culture brothconcentrated 100-fold was treated as indicated or infectious bacteria,as indicated was used to challenge HT29 cells. NF-κB DNA bindingactivity was assayed by EMSA from whole cell extracts prepared 45 minafter treatment. Authenticity of the NF-κB DNA:protein complex wasdetermined using p65(RelA)-specific and p50-specific antibodysupershifts. Panel B: Concentrated Salmonella dublin culture broth (IN)was chromatographed by gel permeation on a SUPEROSE™ 12 (cross-linked,12% agarose-based medium) column. Eluted protein fractions were analyzedby fractionation on 10% SDS-PAGE and visualized by Coomassie blue (CB)staining. Molecular weight markers for chromatography and on the gelsare indicated. Aliquots of each fraction as indicated was used tostimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κBDNA binding activity. Panel C: Concentrated Salmonella dublin culturebroth (IN) was chromatographed by anion exchange chromatography onPOROS® HQ matrix (polystyrenedivinylbenzene particles with quaternizedpolyethyleneimine functional groups). Proteins were eluted with anincreasing NaCl gradient as indicated and analyzed on 10% SDS-PAGE andvisualized by Coomassie blue (CB) staining. Input and aliquots of eachfraction as indicated was used to stimulate HT29 cells and resultantWCEs were analyzed by EMSA for NF-κB DNA binding activity. Elutedmaterial corresponding to protein bands B1-B6 and a blank portion of thegel isolated from a duplicate 10% SDS-PAGE gel, along with buffersamples from the beginning and end NaCl buffer gradient were used tostimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κBDNA binding activity.

FIG. 3 demonstrates that the NF-κB activating factor in Salmonellaculture broth is flagellin, as identified by mass spectrometry.Microcapillary HPLC tandem mass spectrometry of Band 2 digested bytrypsin. Peaks corresponding to Salmonella peptides are numbered andidentified with the corresponding numbered peptide sequence to theright.

FIG. 4 demonstrates that flagellin mutants fail to activate NF-κB. PanelA: EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 minfrom non-infected cells (UN) and after direct infection of HT29 cellswith wild-type E. coli DH5α, wild-type Salmonella dublin or SopE⁻mutant, SopB⁻ mutant, the SopE⁻/SopB⁻ double mutant, wild-typeSalmonella typhimurium strain 1103, the fliC⁻ mutant (fliC::Tn10), thefliC⁻/fljB⁻ double mutant as indicated at an MOI of 50. Panel B: EMSAsassaying for NF-κB DNA binding activity in WCEs prepared 45 min afterchallenge of HT29 cells from non-infected cells (UN) or withsterile-filtered concentrated culture broths from wild-type and mutantbacteria as indicated.

FIG. 5 demonstrates that flagellin is required for activating multiplesignaling pathways during Salmonella infection and leads to nuclearlocalization of NF-κB. Panel A: HT29 cells were left untreated,stimulated with TNFα (10 ng/ml) or a cocktail of anisomycin [An] (20μg/ml)/PMA (12.5 ng/ml) for 15 min, or infected with either wild-type(WT) Salmonella typhimurium strain 1103 or the Salmonella typhimuriumdouble fliC⁻/fljB⁻ mutant strain 134 as indicated. WCE were prepared atthe indicated times or at 10 min for TNF-treated cells or 15 min foranisomycin/PMA treated cells and used in EMSAs to analyze NF-κB DNAbinding activity, or in immuno-kinase assays (KA) using anti-IKK oranti-JNK antibodies to measure IKK and JNK kinase activity on theirrespective substrates GST-IκBα 1-54 and GST-cJun 1-79 (as indicated).Immunoblot (IB) analysis of equivalent amounts (40 μg) of protein fromeach extract was fractionated on SDS-PAGE gels and transferred to PVDFmembranes and probed with the indicated antibodies to detect bulk IKK,JNK, ERK and p38 as indicated. Immunoblot analysis usingphospho-specific antibodies for ERK and p38 to detect activated ERK andp38 are indicated. Panel B: Immunofluorescence demonstrating thatflagellin mutant Salmonella fail to infect HT29 cells and that purifiedflagellin stimulation of HT29 cells leads to NF-κB nuclear p65 (RelA)localization as determined by indirect immunofluorescence. Imaging ofthe treatment indicated HT29 cells grown on coverslips was essentiallythe same as in FIG. 1A & B. False coloring of the DAPI stain was used toenhance the visualization of both DAPI stained nuclei and p65 nuclearlocalization.

FIG. 6 demonstrates that purified flagellin activates signaling pathwaysand proinflammatory gene expression in intestinal epithelial cellsmimicking that of a wild-type Salmonella infection. HT29 cells were leftuntreated or treated with TNFα (10 ng/ml) or a cocktail of anisomycin[An] (20 μg/ml)/PMA (12.5 ng/ml) for 10 min, or with flagellin (1 g/ml)for the indicated times. WCE were prepared and analyzed by EMSA forNF-κB DNA binding activity, immuno-kinase assays (KA) or immunoblotanalysis using phospho-specific antibodies for ERK or p38 to detectactivation and with kinase-specific antibodies as described in FIG. 5Ato detect bulk kinase abundance as indicated. Panel A: EMSA to detectNF-κB DNA binding activity. Panel B: immunoblot and kinase assays todetect IKK, JNK, ERK and p38 kinase activities and protein abundance asin FIG. 5A. Panel C: semi-quantitative RT-PCR of proinflammatory geneexpression of non-treated, wild-type and flagellin double mutantSalmonella typhimurium infected, TNFα (10 ng/ml) or flagellin (1 mg/ml)stimulated cells. HT29 cells were harvested at the indicated times afterthe indicated treatments and isolated RNA was used to make first strandcDNA that subsequently used in RT-PCR reactions using gene-specificprimers for IL1α, IL1β, IL-8, TNFα, MCP1 and β-actin. β-actin was usedas a standard for normalizing expression patterns. Resulting PCRproducts were fractionated on 2% agarose gels and visualized byeithidium bromide staining.

FIG. 7 demonstrates that flagellin-mediated activation of NF-κB is MyD88dependent. Infectious wild-type Salmonella dublin (MOI of 100), IL-1 (20ng/ml), purified flagellin (1 μg/ml) (as indicated), sterile-filteredand concentrated 100 kDa filter retentate supernatant (spt) fromwild-type Salmonella dublin and SopE⁻/SopB⁻ double mutant Salmonelladublin strain SE1SB2 (S2, as indicated) was used to challenge wild-type,MyD88^(−/−) knockout or TLR2^(−/−)/TLR4^(−/−) double knockout MEFs asindicated. WCEs were prepared 45 min after treatments and examined byEMSA to analyze NF-κB DNA binding activity. IL-1 (20 ng/ml) was used asa positive control to monitor MyD88 function.

FIG. 8 demonstrates that TLR5 inhibits flagellin-mediated NF-κB reportergene activity. HT29 cells were transfected in triplicate in 6-welldishes using the indicated DN-TLR mammalian expression vectors orantisense TLR5 (AS TLR5) (2 μg/well), 2×NF-κB Luc reporter gene (100ng/well), pRL-TK Renilla luciferase for normalization (50 ng/well)adjusted to 4 μg total DNA/well with empty vector pcDNA3.1 DNA. Panel A:Fold-induction of 2×NF-κB Luc reporter gene in non-stimulated cells(light shading) and in TNFα (10 ng/ml) treated cells (dark shading).Lysates were prepared 12 h after stimulation. Results of arepresentative experiment are shown. Panel B: HT29 cells transfected asin FIG. 8A were treated with flagellin (1 μg/ml) and cell lysates wereprepared and analyzed as in FIG. 8A. Results of a representativeexperiment are shown.

FIG. 9 demonstrates that flagellin stimulation of intestinal epithelialcells leads to activation of a subset of TLR genes. HT29 cells werestimulated with flagellin (1 mg/ml) and RNA was isolated after 3 h usingTRIZOL™ (mono-phasic solution of phenol and guanidine isothiocyanate)and used to make first strand cDNA. RT-PCR products generated usinggene-specific primers for each TLR as indicated are pictured. β-actinwas used as a standard for normalizing expression patterns.

FIG. 10 demonstrates that TLR5 is expressed in numerous cell types andhas variable responses to flagellin. Panel A: whole cell extracts wereprepared from non-stimulated T84, HT29, A549, HeLa, 293T and T98G cellsand fractionated on a 8% SDS-PAGE gel, proteins were transferred to PVDFmembrane and probed with anti-TLR5 antibody for immunoblot analysis(IB). Protein loading was examined by probing with anti-actin antibody.Panel B: HT29, A549, HeLa, 293T and T98G cells were left untreated (--),treated with flagellin (F) or TNFα (T) and WCEs were prepared after 45min and used in EMSA to monitor NF-κB DNA binding activity. Authenticityof the NF-κB bandshift was tested with supershift of the complex withp65(RelA)-specific antibody.

FIG. 11 demonstrates that p53 deficiency accelerated development of GIsyndrome in mice. Panel A: I.P. injection of PFTα (10 mg/kg) protectsC57Bl/6J mice (if not indicated otherwise, here and below 6-8 weeks oldmales were used) from a single 9 Gy dose of gamma radiation and afractioned cumulative radiation dose 12.5 Gy (5×2.5 Gy). PFTα has noeffect on survival of mice treated with single 12.5 and 25 Gy doses ofIR: (results of representative experiments are shown; Shepherd 4000 CiCesium 137 source at a dose rate of 4 Gy per minute was used). Panel B:Wild-type and p53-null C57Bl/6J mice differ in their relativesensitivity to low (10 Gy) and high (15 Gy) doses of gamma radiation:wild-type mice were more sensitive to 10 Gy but more resistant to 15 Gyas compared to p53-null mice. Panel C: Mice treated with 11 Gy of totalbody gamma irradiation were injected 12 h later with 1.5×10⁷ bone marrowcells from wild type or p53-null syngeneic C57Bl/6J mice. (This dosecauses 100% lethality in nonreconstituted controls group of mice). Twomonths later, after complete recovery of hematopoiesis, animals weretreated with 15 Gy of total body gamma radiation and showed nodifference in death rates between the two groups differing in the p53status of their bone marrow. Panel D: Comparison of dynamics of injuryto small intestines of wild-type and p53-null mice at the indicated timepoints after 15 Gy of gamma radiation indicates accelerated damage inp53-null mice (haematoxylin-eosin stained paraffin sections;magnification ×125). 24 h panels include images of TUNEL staining ifsections of crypts: massive apoptosis is evident in wild type but not inp53-deficient epithelium.

FIG. 12 demonstrates the dynamics of cell proliferation and survival insmall intestine of wild type and p53-null mice. Panel A: Comparison ofproliferation rates in intestines of wild-type and p53 null mice aftertreatment with IR. (Left) Autoradiographs of whole-body sections (1.7×magnification) of 4-week-old wild-type and p53 null mice injectedintraperitoneally with ¹⁴C-thymidine (10 μCi per animal) treated oruntreated with 15 Gy of gamma radiation (Westphal et al 1997). Arrowspoint at intestines. (Right) Comparison of BrdU incorporation in smallintestine of wild-type and p53-null mice at different time points after15 Gy of gamma radiation. BrdU (50 mg/kg) was injected 2 h beforesacrificing mice and immunostaining was done as previously described(Watson & Pritchard 2000). Fragments of 96 h panels are shown at highermagnification (×400). Panel B: Comparison of the number of BrdU positivecells/crypt in small intestine of wild-type and p53-null mice atdifferent time points after 15 Gy of gamma radiation. Three animals wereanalyzed for each time point, five ileum cross sections were preparedfrom each animal and analyzed microscopically to estimate the number ofcrypts and villi. Numbers of BrdU-positive cells in the crypts werecounted in 5 random fields under 200× magnification (100-30 crypts) andthe average number of BrdU-positive cells was plotted. Panel C: Tracingthe number and position of BrdU-labeled cells in small intestine of wildtype and p53-null mice during different time points after 15 Gy of gammaradiation. BrdU was injected 30 min. before irradiation and mice weresacrificed at the indicated time points. Accelerated migration fromcrypts to villi followed by rapid elimination of labeled cells wasobserved in p53-null mice.

FIG. 13 demonstrates that recombinant flagellin is capable of NF-κBactivation.

FIG. 14 shows a representative experiment testing the ability offlagellin to protect mice from radiation. C56BL6 mice (6 week old males,10 animals per group) were injected i.v. with 2.0 μg (0.1 mg/kg) or 5 μg(0.25 mg/kg) of flagellin in PBS. Four hours later, mice were irradiatedwith 15 Gy and mouse survival was monitored daily.

FIG. 15 shows histological sections (HE stained) of small intestinalepithelium of mice that were treated with 15 Gy of gamma radiation withor without i.v. injection of 0.25 mg/kg of flagellin. Completedestruction of crypts and villi in control mouse contrasts with close tonormal morphology of tissue from flagellin-treated animal.

FIG. 16 shows the effect of flagellin on mouse sensitivity to 10 Gy oftotal body gamma radiation.

FIG. 17 shows the effect of flagellin injected i.v. at indicated timesbefore irradiation on mouse sensitivity to 13 Gy (left) and 10 Gy(right) of total body gamma radiation.

FIG. 18 shows the effect of flagellin on mouse sensitivity to 10, 13 and15 Gy of total body gamma radiation.

FIG. 19 shows the domain structure of bacterial flagellin. The Cabackbone trace, hydrophobic core distribution and structural informationof F41. Four distinct hydrophobic cores that de/Ene domains D1, D2a, D2band D3. All the hydrophobic side-chain atoms are displayed with the Cabackbone. Side-chain atoms are color coded: Ala, yellow; Leu, Ile orVal, orange; Phe and Tyr, purple (carbon atoms) and red (oxygen atoms).c, Position and region of various structural features in the amino-acidsequence of flagellin. Shown are, from top to bottom: the F41 fragmentin blue; three b-folium folds in brown; the secondary structuredistribution with a-helix in yellow, b-structure in green, and b-turn inpurple; tic mark at every 50th residue in blue; domains D0, D1, D2 andD3; the axial subunit contact region within the proto-element in cyan;the well-conserved amino-acid sequence in red and variable region inviolet; point mutations in F41 that produce the elements of differentsupercoils. Letters at the bottom indicate the morphology of mutantelements: L (D107E, R124A, R124S, G426A), L-type straight; R (A449V),R-type straight; C (D313Y, A414V, A427V, N433D), curly33. (Samatey etal, Nature 2001).

DETAILED DESCRIPTION

This invention is based on protecting normal cells and tissues fromapoptosis caused by stresses including, but not limited to,chemotherapy, radiation therapy and radiation. There are two majormechanisms controlling apoptosis in the cell: the p53 pathway(pro-apoptotic) and the NF-κB pathway (anti-apoptotic). Both pathwaysare frequently deregulated in tumors: p53 is usually lost, while NF-κBbecomes constitutively active. Hence, inhibition of p53 and activationof NF-κB in normal cells may protect them from death caused by stresses,such as cancer treatment, but would not make tumor cells more resistantto treatment because they have these control mechanisms deregulated.This contradicts the conventional view on p53 and NF-κB, which areconsidered as targets for activation and repression, respectively.

This invention relates to inducing NF-κB activity to protect normalcells from apoptosis. By inducing NF-κB activity in a mammal, normalcells may be protected from apoptosis attributable to cellular stress,which occurs in cancer treatments and hyperthermia; exposure to harmfuldoses of radiation, for example, workers in nuclear power plants, thedefense industry or radiopharmaceutical production, and soldiers; andcell aging. Since NF-κB is constitutively active in many tumor cells,the induction of NF-κB activity may protect normal cells from apoptosiswithout providing a beneficial effect to tumor cells. Once the normalcells are repaired, NF-κB activity may be restored to normal levels.NF-κB activity may be induced to protect such radiation- andchemotherapy-sensitive tissues as the hematopoietic system (includingimmune system), the epithelium of the gut, and hair follicles.

Inducers of NF-κB activity may also be used for several otherapplications. Pathological consequences and death caused by exposure ofmammals to a variety of severe conditions including, but not limited to,radiation, wounding, poisoning, infection, aging, and temperature shock,may result from the activity of normal physiological mechanisms ofstress response, such as induction of programmed cell death (apoptosis)or release of bioactive proteins, cytokines.

Apoptosis normally functions to “clean” tissues from wounded andgenetically damaged cells, while cytokines serve to mobilize the defensesystem of the organism against the pathogen. However, under conditionsof severe injury both stress response mechanisms can by themselves actas causes of death. For example, lethality from radiation may resultfrom massive p53-mediated apoptosis occurring in hematopoietic, immuneand digestive systems. Rational pharmacological regulation of NF-κB mayincrease survival under conditions of severe stress. Control over thesefactors may allow control of both inflammatory response and thelife-death decision of cells from the injured organs.

The protective role of NF-κB is mediated by transcriptional activationof multiple genes coding for: a) anti-apoptotic proteins that block bothmajor apoptotic pathways, b) cytokines and growth factors that induceproliferation and survival of HP and other stem cells, and c) potentROS-scavenging antioxidant proteins, such as MnSOD (SOD-2). Thus, bytemporal activation of NF-κB for radioprotection, it may be possible toachieve not only suppression of apoptosis in cancer patients, but alsothe ability to reduce the rate of secondary cancer incidence because ofsimultaneous immunostimulatory effect, which, may be achieved ifactivation of NF-κB is reached via activation of Toll-like receptors.

Another attractive property of the NF-κB pathway as a target is itsactivation by numerous natural factors that can be considered ascandidate radioprotectants. Among these, are multiplepathogen-associated molecular patterns (PAMPs). PAMPs are molecules thatare not found in the host organism, are characteristic for large groupsof pathogens, and cannot be easily mutated. They are recognized byToll-like receptors (TLRs), the key sensor elements of innate immunity.TLRs act as a first warning mechanism of immune system by inducingmigration and activation of immune cells directly or through cytokinerelease. TLRs are type I membrane proteins, known to work as homo- andheterodimers. Upon ligand binding, TLRs recruit MyD88 protein, anindispensable signaling adaptor for most TLRs. The signaling cascadethat follows leads to effects including (i) activation of NF-κB pathway,and (ii) activation of MAPKs, including Jun N-terminal kinease (JNK).The activation of the NF-κB pathway by Toll-like receptor ligands makesthe ligands attractive as potential radioprotectors. Unlike cytokines,many PAMPs have little effect besides activating TLRs and thus areunlikely to produce side effects. Moreover, many PAMPs are present inhumans.

Consistently with their function of immunocyte activation, all TLRs areexpressed in spleen and peripheral blood leukocytes, with moreTLR-specific patterns of expression in other lymphoid organs and subsetsof leukocytes. However, TLRs are also expressed in other tissues andorgans of the body, e.g., TLR1 is expressed ubiquitously, TLR5 is alsofound in GI epithelium and endothelium, while TLRs 2, 6, 7 and 8 areknown to be expressed in lung.

1. DEFINITIONS

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

As used herein, the terms “administer” when used to describe the dosageof an agent that induces NF-κB activity, means a single dose or multipledoses of the agent.

As used herein, the term “analog”, when used in the context of a peptideor polypeptide, means a peptide or polypeptide comprising one or morenon-standard amino acids or other structural variations from theconventional set of amino acids.

As used herein, the term “antibody” means an antibody of classes IgG,IgM, IgA, IgD or IgE, or fragments or derivatives thereof, includingFab, F(ab′)₂, Fd, and single chain antibodies, diabodies, bispecificantibodies, bifunctional antibodies and derivatives thereof. Theantibody may be a monoclonal antibody, polyclonal antibody, affinitypurified antibody, or mixtures thereof which exhibits sufficient bindingspecificity to a desired epitope or a sequence derived therefrom. Theantibody may also be a chimeric antibody. The antibody may bederivatized by the attachment of one or more chemical, peptide, orpolypeptide moieties known in the art. The antibody may be conjugatedwith a chemical moiety.

As used herein, “apoptosis” refers to a form of cell death that includesprogressive contraction of cell volume with the preservation of theintegrity of cytoplasmic organelles; condensation of chromatin (i.e.,nuclear condensation), as viewed by light or electron microscopy; and/orDNA cleavage into nucleosome-sized fragments, as determined bycentrifuged sedimentation assays. Cell death occurs when the membraneintegrity of the cell is lost (e.g., membrane blebbing) with engulfmentof intact cell fragments (“apoptotic bodies”) by phagocytic cells.

As used herein, the term “cancer” means any condition characterized byresistance to apoptotic stimuli.

As used herein, the term “cancer treatment” means any treatment forcancer known in the art including, but not limited to, chemotherapy andradiation therapy.

As used herein, the term “combination with” when used to describeadministration of an agent that induces NF-κB activity and an additionaltreatment means that the agent may be administered prior to, togetherwith, or after the additional treatment, or a combination thereof.

As used herein, the term “derivative”, when used in the context of apeptide or polypeptide, means a peptide or polypeptide different otherthan in primary structure (amino acids and amino acid analogs). By wayof illustration, derivatives may differ by being glycosylated, one formof post-translational modification. For example, peptides orpolypeptides may exhibit glycosylation patterns due to expression inheterologous systems. If at least one biological activity is retained,then these peptides or polypeptides are derivatives according to theinvention. Other derivatives include, but are not limited to, fusionpeptides or fusion polypeptides having a covalently modified N- orC-terminus, PEGylated peptides or polypeptides, peptides or polypeptidesassociated with lipid moieties, alkylated peptides or polypeptides,peptides or polypeptides linked via an amino acid side-chain functionalgroup to other peptides, polypeptides or chemicals, and additionalmodifications as would be understood in the art.

As used herein, the term “flagellin” means flagellin from any sourceincluding, but not limited to, any bacterial species. The flagellin maybe from a species of Salmonella. Also specifically contemplated arefragments, variants, analogs, homologs, or derivatives of saidflagellin, and combinations thereof. The various fragments, variants,analogs, homologs or derivatives described herein may be 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to awild-type flagellin.

As used herein, the term “fragment”, when used in the context of apeptide or polypeptide, means a peptides of from about 8 to about 50amino acids in length. The fragment may be 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50amino acids in length.

As used herein, the term “homolog”, when used in the context of apeptide or polypeptide, means a peptide or polypeptide sharing a commonevolutionary ancestor.

As used herein, the term “latent TGFβ” means a precursor of TGFβ that isnot in an active form. A latent TGFβ may be a precursor of TGFβcontaining active TGFβ and latency-associated peptide (LAP). A latentTGFβ may also comprise LAP linked to latent TGFβ binding protein. Alatent TGFβ may also be the large latent complex. Furthermore, a latentTGFβ may be a latent TGFβ that is modified so that the rate ofconversion to active TGFβ or ability to be converted to TGFβ has beenreduced. The modified latent TGFβ may be, for example, a TGFβ mutantthat prevents or reduces conversion to active TGFβ.

As used herein, the term “TGFβ” means any isoform of active or latentTGFβ including, but not limited to, TGFβ1, TGFβ2, TGFβ3, TGFβ4 or TGFβ5,and combinations thereof. Also specifically contemplated are fragments,variants, analogs, homologs, or derivatives of said TGFβ isoforms, andcombinations thereof. The various fragments, variants, analogs, homologsor derivatives described herein may be 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a TGFβ isoform.

As used herein, the term “treat” or “treating” when referring toprotection of a mammal from a condition, means preventing, suppressing,repressing, or eliminating the condition. Preventing the conditioninvolves administering a composition of this invention to a mammal priorto onset of the condition. Suppressing the condition involvesadministering a composition of this invention to a mammal afterinduction of the condition but before its clinical appearance.Repressing the condition involves administering a composition of thisinvention to a mammal after clinical appearance of the condition suchthat the condition is reduced or maintained. Elimination the conditioninvolves administering a composition of this invention to a mammal afterclinical appearance of the condition such that the mammal no longersuffers the condition.

As used herein, the term “tumor cell” means any cell characterized byresistance to apoptotic stimuli.

As used herein, the term “variant”, when used in the context of apeptide or polypeptide, means a peptide or polypeptide that differs inamino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. For purposes of this invention, “biological activity”includes, but is not limited to, the ability to be bound by a specificantibody. A conservative substitution of an amino acid, i.e., replacingan amino acid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ∀ 2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporatedherein by reference. Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art. Inone aspect, substitutions are performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

2. METHODS OF TREATMENT a. Constitutively Active NF-κB Tumor

This invention relates to a method of treating a mammal suffering from aconstitutively active NF-κB cancer comprising administering to themammal a composition comprising a therapeutically effective amount of anagent that induces NF-κB activity. The agent that induces NF-κB activitymay be administered in combination with a cancer treatment.

The agent may be administered simultaneously or metronomically withother anti-cancer treatments such as chemotherapy and radiation therapy.The term “simultaneous” or “simultaneously” as used herein, means thatthe other anti-cancer treatment and the compound of This inventionadministered within 48 hours, preferably 24 hours, more preferably 12hours, yet more preferably 6 hours, and most preferably 3 hours or less,of each other. The term “metronomically” as used herein means theadministration of the compounds at times different from the chemotherapyand at certain frequency relative to repeat administration and/or thechemotherapy regiment.

The agent may be administered at any point prior to exposure to thecancer treatment including, but not limited to, about 48 hr, 46 hr, 44hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3hr, 2 hr, or 1 hr prior to exposure. The agent may be administered atany point after exposure to the cancer treatment including, but notlimited to, about 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34hr, 36 hr, 38 hr, 40 hr, 42 hr, 44 hr, 46 hr, or 48 hr after exposure.

The cancer treatment may comprise administration of a cytotoxic agent orcytostatic agent, or combination thereof. Cytotoxic agents preventcancer cells from multiplying by: (1) interfering with the cell'sability to replicate DNA and (2) inducing cell death and/or apoptosis inthe cancer cells. Cytostatic agents act via modulating, interfering orinhibiting the processes of cellular signal transduction which regulatecell proliferation and sometimes at low continuous levels.

Classes of compounds that may be used as cytotoxic agents include thefollowing: alkylating agents (including, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes): uracil mustard, chlormethine, cyclophosphamide (CYTOXAN®),ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine,triethylenethiophosphoramine, busulfan, carmustine, lomustine,streptozocin, dacarbazine, and temozolomide; antimetabolites (including,without limitation, folic acid antagonists, pyrimidine analogs, purineanalogs and adenosine deaminase inhibitors): methotrexate,5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, pentostatine, and gemcitabine;natural products and their derivatives (for example, vinca alkaloids,antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins):vinblastine, vincristine, vindesine, bleomycin, dactinomycin,daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c, paclitaxel(paclitaxel is commercially available as TAXOL®), mithramycin,deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons (preferablyIFN-α), etoposide, and teniposide.

Other proliferative cytotoxic agents are navelbene, CPT-11, anastrazole,letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, anddroloxafine.

Microtubule affecting agents interfere with cellular mitosis and arewell known in the art for their cytotoxic activity. Microtubuleaffecting agents useful in the invention include, but are not limitedto, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(TAXOL®, NSC 125973), TAXOL® derivatives (e.g., derivatives (e.g., NSC608832), thiocolchicine NSC 361792), trityl cysteine (NSC 83265),vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574),natural and synthetic epothilones including but not limited toepothilone A, epothilone B, and discodermolide (see Service, (1996)Science, 274:2009) estramustine, nocodazole, MAP4, and the like.Examples of such agents are also described in Bulinski (1997) J. CellSci. 110:3055 3064; Panda (1997) Proc. Natl. Acad. Sci. USA94:10560-10564; Muhlradt (1997) Cancer Res. 57:3344-3346; Nicolaou(1997) Nature 387:268-272; Vasquez (1997) Mol. Biol. Cell. 8:973-985;and Panda (1996) J. Biol. Chem 271:29807-29812.

Also suitable are cytotoxic agents such as epidophyllotoxin; anantineoplastic enzyme; a topoisomerase inhibitor; procarbazine;mitoxantrone; platinum coordination complexes such as cis-platin andcarboplatin; biological response modifiers; growth inhibitors;antihormonal therapeutic agents; leucovorin; tegafur; and haematopoieticgrowth factors.

Cytostatic agents that may be used include, but are not limited to,hormones and steroids (including synthetic analogs):17.alpha.-ethinylestradiol, diethylstilbestrol, testosterone,prednisone, fluoxymesterone, dromostanolone propionate, testolactone,megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone,triamcinolone, hlorotrianisene, hydroxyprogesterone, aminoglutethimide,estramustine, medroxyprogesteroneacetate, leuprolide, flutamide,toremifene, zoladex.

Other cytostatic agents are antiangiogenics such as matrixmetalloproteinase inhibitors, and other VEGF inhibitors, such asanti-VEGF antibodies and small molecules such as ZD6474 and SU6668 arealso included. Anti-Her2 antibodies from Genetech may also be utilized.A suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Alsoincluded are Imclone antibody C225 immunospecific for the EGFR, and srcinhibitors.

Also suitable for use as an cytostatic agent is CASODEX® (bicalutamide,Astra Zeneca) which renders androgen-dependent carcinomasnon-proliferative. Yet another example of a cytostatic agent is theantiestrogen tamoxifen which inhibits the proliferation or growth ofestrogen dependent breast cancer. Inhibitors of the transduction ofcellular proliferative signals are cytostatic agents. Representativeexamples include epidermal growth factor inhibitors, Her-2 inhibitors,MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Srckinase inhibitors, and PDGF inhibitors.

A variety of cancers may be treated according to this inventionincluding, but not limited to, the following: carcinoma including thatof the bladder (including accelerated and metastatic bladder cancer),breast, colon (including colorectal cancer), kidney, liver, lung(including small and non-small cell lung cancer and lungadenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphaticsystem, rectum, larynx, pancreas (including exocrine pancreaticcarcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin(including squamous cell carcinoma); hematopoietic tumors of lymphoidlineage including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocyticlymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineageincluding acute and chronic myelogenous leukemias, myelodysplasticsyndrome, myeloid leukemia, and promyelocytic leukemia; tumors of thecentral and peripheral nervous system including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal originincluding fibrosarcoma, rhabdomyoscarcoma, and osteosarcoma; and othertumors including melanoma, xenoderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer, and teratocarcinoma. In a preferredembodiment, this invention is used to treat cancers of gastrointestinaltract.

b. Treatment of Side Effects from Cancer Treatment

This invention also relates to a method of treating a mammal sufferingfrom damage to normal tissue attributable to treatment of aconstitutively active NF-κB cancer, comprising administering to themammal a composition comprising a therapeutically effective amount of anagent that induces NF-κB activity. The agent that induces NF-κB activitymay be administered in combination with a cancer treatment describedabove.

c. Modulation of Cell Aging

This invention also relates to a method of modulating cell aging in amammal, comprising administering to the mammal a therapeuticallyeffective amount of an agent that induces NF-κB activity. The agent thatinduces NF-κB activity may be administered in combination with othertreatments.

The agent may be administered at any point prior to administration ofthe other treatment including, but not limited to, about 48 hr, 46 hr,44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3hr, 2 hr, or 1 hr prior to administration. The agent may be administeredat any point after administration of the other treatment including, butnot limited to, about 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr,14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34hr, 36 hr, 38 hr, 40 hr, 42 hr, 44 hr, 46 hr, or 48 hr afteradministration.

d. Treatment of Stress

This invention also relates to a method of treating a mammal sufferingfrom damage to normal tissue attributable to stress, comprisingadministering to the mammal a composition comprising a therapeuticallyeffective amount of an agent that induces NF-κB activity. The agent thatinduces NF-κB activity may be administered in combination with othertreatments. The stress may be attributable to any source including, butnot limited to, radiation, wounding, poisoning, infection, andtemperature shock.

The composition comprising an inducer of NF-κB may be administered atany point prior to exposure to the stress including, but not limited to,about 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, or 1 hr prior to exposure. Thecomposition comprising an inducer of NF-κB may be administered at anypoint after exposure to the stress including, but not limited to, about1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr,20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40hr, 42 hr, 44 hr, 46 hr, or 48 hr after exposure.

e. Radiation

This invention is also related to the protection of cells from theeffects of exposure to radiation. Injury and death of normal cells fromionizing radiation is a combination of a direct radiation-induced damageto the exposed cells and an active genetically programmed cell reactionto radiation-induced stress resulting in a suicidal death or apoptosis.Apoptosis plays a key role in massive cell loss occurring in severalradiosensitive organs (i.e., hematopoietic and immune systems,epithelium of digestive tract, etc.), the failure of which determinesgeneral radiosensitivity of the organism.

Exposure to ionizing radiation (IR) may be short- or long-term, it maybe applied as a single or multiple doses, to the whole body or locally.Thus, nuclear accidents or military attacks may involve exposure to asingle high dose of whole body irradiation (sometimes followed by along-term poisoning with radioactive isotopes). The same is true (withstrict control of the applied dose) for pretreatment of patients forbone marrow transplantation when it is necessary to preparehematopoietic organs for donor's bone marrow by “cleaning” them from thehost blood precursors. Cancer treatment may involve multiple doses oflocal irradiation that greatly exceeds lethal dose if it were applied asa total body irradiation. Poisoning or treatment with radioactiveisotopes results in a long-term local exposure to radiation of targetedorgans (e.g., thyroid gland in the case of inhalation of 125I). Finally,there are many physical forms of ionizing radiation differingsignificantly in the severity of biological effects.

At the molecular and cellular level, radiation particles are able toproduce breakage and cross-linking in the DNA, proteins, cell membranesand other macromolecular structures. Ionizing radiation also induces thesecondary damage to the cellular components by giving rise to the freeradicals and reactive oxygen species (ROS). Multiple repair systemscounteract this damage, such as several DNA repair pathways that restorethe integrity and fidelity of the DNA, and antioxidant chemicals andenzymes that scavenge the free radicals and ROS and reduce the oxidizedproteins and lipids. Cellular checkpoint systems detect the DNA defectsand delay cell cycle progression until damage is repaired or decision tocommit cell to growth arrest or programmed cell death (apoptosis) isreached

Radiation can cause damage to mammalian organism ranging from mildmutagenic and carcinogenic effects of low doses to almost instantkilling by high doses. Overall radiosensitivity of the organism isdetermined by pathological alterations developed in several sensitivetissues that include hematopoietic system, reproductive system anddifferent epithelia with high rate of cell turnover.

Acute pathological outcome of gamma irradiation leading to death isdifferent for different doses and is determined by the failure ofcertain organs that define the threshold of organism's sensitivity toeach particular dose. Thus, lethality at lower doses occurs from bonemarrow aplasia, while moderate doses kill faster by inducing agastrointestinal (GI) syndrome. Very high doses of radiation can causealmost instant death eliciting neuronal degeneration.

Organisms that survive a period of acute toxicity of radiation cansuffer from long-term remote consequences that include radiation-inducedcarcinogenesis and fibrosis developing in exposed organs (e.g., kidney,liver or lungs) months and years after irradiation.

Cellular DNA is the major target of IR that causes a variety of types ofDNA damage (genotoxic stress) by direct and indirect (freeradical-based) mechanisms. All organisms maintain DNA repair systemcapable of effective recovery of radiation-damaged DNA; errors in DNArepair process may lead to mutations.

Tumors are generally more sensitive to gamma radiation and can betreated with multiple local doses that cause relatively low damage tonormal tissue. Nevertheless, in some instances, damage of normal tissuesis a limiting factor in application of gamma radiation for cancertreatment. The use of gamma-irradiation during cancer therapy byconventional, three-dimensional conformal or even more focused BeamCathdelivery has also dose-limiting toxicities caused by cumulative effectof irradiation and inducing the damage of the stem cells of rapidlyrenewing normal tissues, such as bone marrow and gastrointestinal (GI)tract.

At high doses, radiation-induced lethality is associated with so-calledhematopoietic and gastrointestinal radiation syndromes. Hematopoieticsyndrome is characterized by loss of hematopoietic cells and theirprogenitors making it impossible to regenerate blood and lymphoidsystem. The death usually occurs as a consequence of infection (resultof immunosuppression), hemorrhage and/or anemia. GI syndrome is causedby massive cell death in the intestinal epithelium, predominantly in thesmall intestine, followed by disintegration of intestinal wall and deathfrom bacteriemia and sepsis. Hematopoietic syndrome usually prevails atthe lower doses of radiation and leads to the more delayed death than GIsyndrome.

In the past, radioprotectants were typically antioxidants—both syntheticand natural. More recently, cytokines and growth factors have been addedto the list of radioprotectants; the mechanism of their radioprotectionis considered to be a result of facilitating effect on regeneration ofsensitive tissues. There is no clear functional distinction between bothgroups of radioprotectants, however, since some cytokines induce theexpression of the cellular antioxidant proteins, such as manganesesuperoxide dismutase (MnSOD) and metallothionein.

The measure of protection for a particular agent is expressed by dosemodification factor (DMF or DRF). DMF is determined by irradiating theradioprotector treated subject and untreated control subjects with arange of radiation doses and then comparing the survival or some otherendpoints. DMF is commonly calculated for 30-day survival (LD50/30drug-treated divided by LD50/30 vehicle-treated) and quantifies theprotection of the hematopoietic system. In order to estimategastrointestinal system protection, LD50 and DMF are calculated for 6-or 7-day survival. DMF values provided herein are 30-day unlessindicated otherwise.

Inducers of NF-κB possess strong pro-survival activity at the cellularlevel and on the organism as a whole. In response to super-lethal dosesof radiation, inducers of NF-κB inhibit both gastrointestinal andhematopoietic syndromes, which are the major causes of death from acuteradiation exposure. As a result of these properties, inducers of NF-κBmay be used to treat the effects of natural radiation events and nuclearaccidents. Moreover, since inducers of NF-κB acts through mechanismsdifferent from all presently known radioprotectants, they can be used incombination with other radioprotectants, thereby, dramaticallyincreasing the scale of protection from ionizing radiation.

As opposed to conventional radioprotective agents (e.g., scavengers offree radicals), anti-apoptotic agents may not reduce primaryradiation-mediated damage but may act against secondary events involvingactive cell reaction on primary damage, therefore complementing theexisting lines of defense. Pifithrin-alpha, a pharmacological inhibitorof p53 (a key mediator of radiation response in mammalian cells), is anexample of this new class of radioprotectants. However, the activity ofp53 inhibitors is limited to protection of the hematopoietic system andhas no protective effect in digestive tract (gastrointestinal syndrome),therefore, reducing therapeutic value of these compounds. Anti-apoptoticpharmaceuticals with broader range of activity are desperately needed.

Inducers of NF-κB may be used as a radioprotective agent to extend therange of tolerable radiation doses by increasing radioresistance ofhuman organism beyond the levels achievable by currently availablemeasures (shielding and application of existing bioprotective agents)and drastically increase the chances of crew survival in case of onboardnuclear accidents or large-scale solar particle events. With anapproximate DMF (30-day survival) greater than 1.5, the NF-κB inducerflagellin is more effective than any currently reported naturalcompound.

Inducers of NF-κB are also useful for treating irreplaceable cell losscaused by low-dose irradiation, for example, in the central nervoussystem and reproductive organs. Inducers of NF-κB may also be usedduring cancer chemotherapy to treat the side effects associated withchemotherapy, including alopecia.

In one embodiment, a mammal is treated for exposure to radiation,comprising administering to the mammal a composition comprising atherapeutically effective amount of a composition comprising an inducerof NF-κB. The composition comprising an inducer of NF-κB may beadministered in combination with one or more radioprotectants. The oneor more radioprotectants may be any agent that treats the effects ofradiation exposure including, but not limited to, antioxidants, freeradical scavengers and cytokines.

Inducers of NF-κB may inhibit radiation-induced programmed cell death inresponse to damage in DNA and other cellular structures; however,inducers of NF-κB may not deal with damage at the cellular and may notprevent mutations. Free radicals and reactive oxygen species (ROS) arethe major cause of mutations and other intracellular damage.Antioxidants and free radical scavengers are effective at preventingdamage by free radicals. The combination of an inducer of NF-κB and anantioxidant or free radical scavenger may result in less extensiveinjury, higher survival, and improved health for mammal exposed toradiation. Antioxidants and free radical scavengers that may be used inthe practice of the invention include, but are not limited to, thiols,such as cysteine, cysteamine, glutathione and bilirubin; amifostine(WR-2721); vitamin A; vitamin C; vitamin E; and flavonoids such asIndian holy basil (Ocimum sanctum), orientin and vicenin.

Inducers of NF-κB may also be administered in combination with a numberof cytokines and growth factors that confer radioprotection byreplenishing and/or protecting the radiosensitive stem cell populations.Radioprotection with minimal side effects may be achieved by the use ofstem cell factor (SCF, c-kit ligand), Flt-3 ligand, and interleukin-1fragment IL-1b-rd. Protection may be achieved through induction ofproliferation of stem cells (all mentioned cytokines), and prevention oftheir apoptosis (SCF). The treatment allows accumulation of leukocytesand their precursors prior to irradiation thus enabling quickerreconstitution of the immune system after irradiation. SCF efficientlyrescues lethally irradiated mice with DMF in range 1.3-1.35 and is alsoeffective against gastrointestinal syndrome. Flt-3 ligand also providesstrong protection in mice (70-80% 30-day survival at LD100/30,equivalent to DMF>1.2) and rabbits (15, 16).

Several factors, while not cytokines by nature, stimulate theproliferation of the immunocytes and may be used in combination withinducers of NF-κB. 5-AED (5-androstenediol) is a steroid that stimulatesthe expression of cytokines and increases resistance to bacterial andviral infections. A subcutaneous injection of 5-AED in mice 24 h beforeirradiation improved survival with DMF=1.26. Synthetic compounds, suchas ammonium tri-chloro(dioxoethylene-O,O′-) tellurate (AS-101), may alsobe used to induce secretion of numerous cytokines and for combinationwith inducers of NF-κB.

Growth factors and cytokines may also be used to provide protectionagainst the gastrointestinal syndrome. Keratinocyte growth factor (KGF)promotes proliferation and differentiation in the intestinal mucosa, andincreases the post-irradiation cell survival in the intestinal crypts.Hematopoietic cytokine and radioprotectant SCF may also increaseintestinal stem cell survival and associated short-term organismsurvival.

Inducers of NF-κB may offer protection against both gastrointestinal(GI) and hematopoietic syndromes. Since mice exposed to 15 Gy ofwhole-body lethal irradiation died mostly from GI syndrome, acomposition comprising an inducer of NF-κB and one or more inhibitors ofGI syndrome may be more effective. Inhibitors of GI syndrome that may beused in the practice of the invention include, but are not limited to,cytokines such as SCF and KGF.

The composition comprising an inducer of NF-κB may be administered atany point prior to exposure to radiation including, but not limited to,about 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, or 1 hr prior to exposure. Thecomposition comprising an inducer of NF-κB may be administered at anypoint after exposure to radiation including, but not limited to, about 1hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40hr, 42 hr, 44 hr, 46 hr, or 48 hr after exposure to radiation.

3. AGENT

This invention also relates to an agent that induces NF-κB activity. Theagent may be an artificially synthesized compound or a naturallyoccurring compound. The agent may be a low molecular weight compound,polypeptide or peptide, or a fragment, analog, homolog, variant orderivative thereof.

The agent may also be an NF-κB inducing cytokine including, but notlimited to, IL2, IL6, TNF and TGFβ. The agent may also be aprostaglandin. The agent may also be a growth factor including, but notlimited to, KGF and PDGF. The agent may also be an antibody that inducesNF-κB activity.

a. Flagellin

In one embodiment, the agent is flagellin, which may be from a bacteriaincluding, but not limited to, a species of Salmonella, such as S.typhimurium. As shown in the Examples below, flagellin possesses strongpro-survival activity at the cellular level and on the organism as awhole.

A fragment, variant, analog, homolog, or derivative of an inducer ofNF-κB, such as flagellin, with beneficial properties may be obtained byrational-based design based on the domain structure of flagellin. Thedomain structure of Salmonella flagellin is described in the literature(FIG. 19). Flagellin has conserved domains (D1 and D2) at the N terminusand C terminus and a middle hypervariable domain (D3) (Samatey, et al2001, Eaves-Pyles T, et al 2001a). Results with a recombinant proteincontaining the amino D1 and D2 and carboxyl D1 and D2 separated by anEscherichia coli hinge (ND 1-2/ECH/CD2) indicate that D1 and D2 arebioactive when coupled to an ECH element. This chimera, but not thehinge alone, induced I_(κ)B_(α) degradation, NF-κB activation, and NOand IL-8 production in two intestinal epithelial cell lines. Thenon-conserved D3 domain is on the surface of the flagellar filament andcontains the major antigenic epitopes. The potent proinflammatoryactivity of flagellin may reside in the highly conserved N and C D1 andD2 regions.

b. Parasitic Inducers of NF-κB

The properties of flagellin suggest that additional modulators of NF-κBmay be found in parasites. There are a number of parasites that dependon the repression of apoptosis since they cannot survive without thecells of the host. These organisms may have adapted for effectivepersistence in the host organism by secreting anti-apoptotic factors.Like advanced tumors, these organisms secrete factors may be capable ofincreasing their own survival and resolving their conflict with thestress response defensive mechanism of the host.

Anti-apoptotic factors from parasitic or symbiotic organisms have passedthrough millions of years of adaptation to minimize harm on the hostorganism that would affect viability. As a result, these factors mayrequire little, if any, additional modifications and may be useddirectly as they are or with minimal modifications. The factors may beuseful to treat stress-mediated apoptosis, such as side effectsassociated with chemo- and radiation therapy.

This invention is also related to methods for screening parasites foridentifying modulators of NF-κB. The candidate modulators may be fromparasites of humans or non-human primates. The parasites are preferablyextracellular parasites of the host. The parasites may also besymbionts. Parasites from which modulators of This invention may beisolated include, but are not limited to, Mycoplasma, Chlamydia andSalmonella. These modulators may be identified using the screeningmethods described herein, as well as by biochemical and geneticselection approaches, in vitro testing, cell death protecting agents,and in vivo.

4. COMPOSITION

This invention also relates to a composition comprising atherapeutically effective amount of an inducer of NF-κB. The compositionmay be a pharmaceutical composition, which may be produced using methodswell known in the art. As described above, the composition comprising aninducer of NF-κB may be administered to a mammal for the treatment ofconditions associated with apoptosis including, but not limited to,exposure to radiation, side effect from cancer treatments, stress andcell aging. The composition may also comprise additional agentsincluding, but not limited to, a radioprotectant or a chemotherapeuticdrug.

a. Administration

Compositions of this invention may be administered in any mannerincluding, but not limited to, orally, parenterally, sublingually,transdermally, rectally, transmucosally, topically, via inhalation, viabuccal administration, or combinations thereof. Parenteraladministration includes, but is not limited to, intravenous,intraarterial, intraperitoneal, subcutaneous, intramuscular,intrathecal, and intraarticular. For veterinary use, the composition maybe administered as a suitably acceptable formulation in accordance withnormal veterinary practice. The veterinarian can readily determine thedosing regimen and route of administration that is most appropriate fora particular animal.

b. Formulation

Compositions of this invention may be in the form of tablets or lozengesformulated in a conventional manner. For example, tablets and capsulesfor oral administration may contain conventional excipients including,but not limited to, binding agents, fillers, lubricants, disintegrantsand wetting agents. Binding agents include, but are not limited to,syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch andpolyvinylpyrrolidone. Fillers include, but are not limited to, lactose,sugar, microcrystalline cellulose, maizestarch, calcium phosphate, andsorbitol. Lubricants include, but are not limited to, magnesiumstearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants include, but are not limited to, potato starch and sodiumstarch glycollate. Wetting agents include, but are not limited to,sodium lauryl sulfate). Tablets may be coated according to methods wellknown in the art.

Compositions of this invention may also be liquid formulationsincluding, but not limited to, aqueous or oily suspensions, solutions,emulsions, syrups, and elixirs. The compositions may also be formulatedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may contain additives including,but not limited to, suspending agents, emulsifying agents, nonaqueousvehicles and preservatives. Suspending agent include, but are notlimited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup,gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel, and hydrogenated edible fats. Emulsifying agents include,but are not limited to, lecithin, sorbitan monooleate, and acacia.Nonaqueous vehicles include, but are not limited to, edible oils, almondoil, fractionated coconut oil, oily esters, propylene glycol, and ethylalcohol. Preservatives include, but are not limited to, methyl or propylp-hydroxybenzoate and sorbic acid.

Compositions of this invention may also be formulated as suppositories,which may contain suppository bases including, but not limited to, cocoabutter or glycerides. Compositions of this invention may also beformulated for inhalation, which may be in a form including, but notlimited to, a solution, suspension, or emulsion that may be administeredas a dry powder or in the form of an aerosol using a propellant, such asdichlorodifluoromethane or trichlorofluoromethane. Compositions of thisinvention may also be formulated transdermal formulations comprisingaqueous or nonaqueous vehicles including, but not limited to, creams,ointments, lotions, pastes, medicated plaster, patch, or membrane.

Compositions of this invention may also be formulated for parenteraladministration including, but not limited to, by injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The composition may also be providedin a powder form for reconstitution with a suitable vehicle including,but not limited to, sterile, pyrogen-free water.

Compositions of this invention may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The compositions may be formulated withsuitable polymeric or hydrophobic materials (as an emulsion in anacceptable oil, for example), ion exchange resins, or as sparinglysoluble derivatives (as a sparingly soluble salt, for example).

c. Dosage

A therapeutically effective amount of the agent required for use intherapy varies with the nature of the condition being treated, thelength of time that induction of NF-κB activity is desired, and the ageand the condition of the patient, and is ultimately determined by theattendant physician. In general, however, doses employed for adult humantreatment typically are in the range of 0.001 mg/kg to about 200 mg/kgper day. The dose may be about 1 μg/kg to about 100 μg/kg per day. Thedesired dose may be conveniently administered in a single dose, or asmultiple doses administered at appropriate intervals, for example astwo, three, four or more subdoses per day. Multiple doses often aredesired, or required, because NF-κB activity in normal cells may bedecreased once the agent is no longer administered.

The dosage of an inducer of NF-κB may be at any dosage including, butnot limited to, about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg,125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg,450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg,775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925μg/kg, 950 μg/kg, 975 μg/kg or 1 mg/kg.

5. SCREENING METHODS

This invention also relates to methods of identifying agents that induceNF-κB activity. An agent that induces NF-κB activity may be identifiedby a method comprising adding a suspected inducer of NF-κB activity toan NF-κB activated expression system, comparing the level of NF-κBactivated expression to a control, whereby an inducer of NF-κB activityis identified by the ability to increase the level of NF-κB activatedexpression system.

Candidate agents may be present within a library (i.e., a collection ofcompounds). Such agents may, for example, be encoded by DNA moleculeswithin an expression library. Candidate agent be present in conditionedmedia or in cell extracts. Other such agents include compounds known inthe art as “small molecules,” which have molecular weights less than 10⁵daltons, preferably less than 10⁴ daltons and still more preferably lessthan 10³ daltons. Such candidate agents may be provided as members of acombinatorial library, which includes synthetic agents (e.g., peptides)prepared according to multiple predetermined chemical reactions. Thosehaving ordinary skill in the art will appreciate that a diverseassortment of such libraries may be prepared according to establishedprocedures, and members of a library of candidate agents can besimultaneously or sequentially screened as described herein.

The screening methods may be performed in a variety of formats,including in vitro, cell-based and in vivo assays. Any cells may be usedwith cell-based assays. Preferably, cells for use with this inventioninclude mammalian cells, more preferably human and non-human primatecells. Cell-base screening may be performed using genetically modifiedtumor cells expressing surrogate markers for activation of NF-κB. Suchmarkers include, but are not limited to, bacterial beta-galactosidase,luciferase and enhanced green fluorescent protein (EGFP). The amount ofexpression of the surrogate marker may be measured using techniquesstandard in the art including, but not limited to, colorimetery,luminometery and fluorimetery.

The conditions under which a suspected modulator is added to a cell,such as by mixing, are conditions in which the cell can undergoapoptosis or signaling if essentially no other regulatory compounds arepresent that would interfere with apoptosis or signaling. Effectiveconditions include, but are not limited to, appropriate medium,temperature, pH and oxygen conditions that permit cell growth. Anappropriate medium is typically a solid or liquid medium comprisinggrowth factors and assimilable carbon, nitrogen and phosphate sources,as well as appropriate salts, minerals, metals and other nutrients, suchas vitamins, and includes an effective medium in which the cell can becultured such that the cell can exhibit apoptosis or signaling. Forexample, for a mammalian cell, the media may comprise Dulbecco'smodified Eagle's medium containing 10% fetal calf serum.

Cells may be cultured in a variety of containers including, but notlimited to tissue culture flasks, test tubes, microtiter dishes, andpetri plates. Culturing is carried out at a temperature, pH and carbondioxide content appropriate for the cell. Such culturing conditions arealso within the skill in the art.

Methods for adding a suspected modulator to the cell includeelectroporation, microinjection, cellular expression (i.e., using anexpression system including naked nucleic acid molecules, recombinantvirus, retrovirus expression vectors and adenovirus expression), use ofion pairing agents and use of detergents for cell permeabilization.

This invention has multiple aspects, illustrated by the followingnon-limiting examples.

EXAMPLE 1 P53 Deficiency Accelerated Development of GI Syndrome in Mice

Primary cause of death from ionizing radiation (IR) of mammals dependson the radiation dose. At doses of up to 9-10 Gy, mice die 12-20 dayslater, primarily from lethal bone marrow depletion-hematopoietic (HP)syndrome. At this dose, irradiated mice can be rescued from lethality bybone marrow transplantation. Animals that received >15 Gy die between7-12 days after treatment (before hematopoietic syndrome could killthem) from complications of damage to the smallintestine-gastrointestinal (GI) syndrome (Gudkov & Komarova 2003). Inboth cases of HP and GI syndromes, lethal damage of tissues starts frommassive p53 dependent apoptosis (Potten 1992, Merritt 1994, Cui et al1995, Potten et al 1994), the observation that allowed us earlier tosuggest that p53 could be a determinant of radiation-induced death.Consistently, p53-deficient mice are resistant to doses of radiationthat kill through HP syndrome (Westphal et al 1997, Komarov et al 1999),and lethality of wild type animals receiving 6-11 Gy of gamma radiationcan be reduced by temporary pharmacological inhibition of p53 by smallmolecule p53 inhibitor pifithrin-alpha (PFT) (Komarov et al 1999).Definition of p53 as a factor sensitizing tissues to genotoxic stresswas further strengthened by demonstrating the p53 dependence of hairloss (alopecia) occurring as a result of experimental chemotherapy orradiation (Botchkarev et al 2000). Hence, based on previousobservations, one could expect that p53 continues to play an importantrole in development of lethal GI syndrome after higher doses of IR.Surprisingly, p53-deficiency sensitizes mice to higher doses of IRcausing lethal gastro-intestinal syndrome (FIG. 11). Continuous cellproliferation in the crypts of p53-deficient epithelium after IRcorrelates with accelerated death of damaged cells of crypt and rapiddestruction of villi. p53 prolongs survival by inducing growth arrest inthe crypts of small intestine thereby preserving integrity of the guts(FIG. 12). Thus, proapoptotic function of p53 promotes hematopoieticsyndrome while its growth arrest function delays development ofgastro-intestinal syndrome.

The dynamics of cell population in the small intestine has been analyzedin great detail. Cell proliferation in epithelia of the guts is limitedto the crypts where stem cells and early proliferating progenitors arelocated. After a couple of cell divisions, already differentiateddescendants of crypt stem cells move up the villi to be shed at thevillar tip. In the small intestine of the mouse, the entire “trip” ofthe cell (the proliferative compartment to the tip of the villus)normally takes between 3 and 5 days (Potten 1992, Potten et al 1997,Booth et al 2002, Somosy et al 2002). Although reaction of the smallintestine to gamma radiation has been well examined at apathomorphological level, it still remains unclear what is the exactcause of GI lethality, including the primary event. Death may occur as adirect consequence of the damage of epithelial crypt cells and followeddenudation of villi leading to fluid and electrolyte imbalance,bacteremia and endotoxemia. Besides inflammation and stromal responses,endothelial dysfunctions seem to be the important factors contributingto lethality (Potten et al 1997, Somosy et al 2002). In summary,pharmacological suppression of p53 that was shown to be so effective asa method of protection from IR-induced HP syndrome, is useless (if notdetrimental) against GI syndrome. Therefore, it is necessary to developalternative approaches to radioprotection of epithelium of smallintestine that will rely on another mechanism, such as, for example,activation of NF-κB and subsequent inhibition of cell death.

EXAMPLE 2 Salmonella Infection Activates NF-κB

Salmonella infection leads to potent IKK and NF-κB activation andactivation of the proinflammatory gene program (Elewaut et al 1999).Previous studies suggest that about 30-40% of intestinal epithelialcells are infected during a typical Salmonella infection in culturedintestinal epithelial cells (Valdivia et al 1996). We wished to addressthe question of how bacterial infection of about 30% of the host cellscould give rise to NF-κB DNA binding activity equivalent to activationof NF-κB in nearly all of the host cells as TNFα treatment does.

To examine this phenomenon in detail, HT29 cells were eithermock-infected or infected at a MOI of 50 for one hour with wild-type S.typhimurium that had been transformed with the plasmid pFM10.1 thatencodes green fluorescent protein (GFP) under the control of theSalmonella ssaH promoter and only functions once the bacteria hasinvaded the host cell (Valdivia et al. 1997). Cells that were invaded bySalmonella were detected by direct fluorescence microscopy of GFPexpression. p65(RelA) localization was monitored by indirectimmunoflourescence of rabbit anti-p65 antibody detected withFITC-conjugated donkey anti-rabbit antibody. DAPI was used to stainnuclei.

As can be seen in FIG. 1A, GFP expression occurs in about thirty toforty percent of the cells. We next examined the localization of theNF-κB subunit p65 (RelA) in non-treated (mock-infected), Salmonellainfected or TNFα (10 ng/ml) stimulated cells. P65 (RelA) was localizedto the cytoplasm in non-treated cells, whereas p65 (RelA) was localizedto the nucleus in Salmonella infected cells or in TNFα treated cells(FIG. 1B). These results demonstrate that Salmonella infection activatesNF-κB in virtually all of the cells even though only a minority of thembecome infected.

EXAMPLE 3 Flagellin Activates NF-κB

Since Salmonella infection of intestinal epithelial cells in culture ledto only roughly 30% infection but activation of NF-κB in nearly all ofthe cells, we anticipated that NF-κB activation was in response to hostcell recognition of bacteria structural components or products producedby the bacteria and not by the invasion process. Invasion itself hasbeen demonstrated not to be required for activation of theproinflamatory gene program as had previously been thought (16). Toinvestigate this possibility sterile-filtered S. dublin culture brothleft either untreated or boiled for twenty minutes was used to challengeHT29 intestinal epithelial cells and NF-κB DNA binding activity wasmonitored by electromobility shift assays (EMSAs) of whole cell extracts(WCE) prepared forty-five minutes after exposure (3, 40). Potentactivation of NF-κB in response to the broth under both conditions wasobserved indicating the activating factor was heat-stable.

The native sterile-filtered concentrated broth was subsequently treatedwith DNase, RNase, proteinase K or crudely size fractionated on 100 kDacentricon filters. The variously treated broths were then used tochallenge HT29 intestinal epithelial cells and WCEs were prepared afterforty-five minutes and NF-κB DNA binding activity was analyzed by EMSA(FIG. 2A). Direct infection of HT29 cells by S. typhimurium 1103 orexposure to the culture broths (supt), as indicated, induced NF-κB DNAbinding activity, while the activity-inducing factor was found to besensitive to protease digestion and was retained by a 100 kDa filter(FIG. 2A). To further determine the identity of the NF-κB inducingactivity, sterile-filtered concentrated broth culture was fractionatedby SUPEROSE™ 12 (cross-linked, 12% agarose-based medium gel permeationchromatography (FIG. 2B) and by anion exchange chromatography (FIG. 2C).Aliquots of chromatography fractions were assayed for their ability toactivate NF-κB in HT29 cells and analyzed by EMSA. As can be seen fromthe Coomassie blue stained gel (FIG. 2B, top panel), increased NF-κB DNAbinding activity (FIG. 2B, lower panel lanes 4-6) corresponded to theincreased abundance of an approximately 55 kDa protein. Anion exchangechromatography on POROS® HQ matrix (polystyrenedivinylbenzene particleswith quaternized polyethyleneimide functional groups) and elution ofbound proteins with an increasing salt gradient as indicated (FIG. 2C)demonstrated that NF-κB DNA binding-inducing activity corresponded tochromatographic fractions containing an increased abundance of the 55kDa protein (FIG. 2C top panel, and data not shown). Eluted fractionsobserved in FIG. 2C were concentrated and fractionated on preparative12% SDS-PAGE gels and bands corresponding to B1-B6 were cut from thegels and the proteins eluted, precipitated, renatured, and used tostimulate HT29 cells. Whole cell extracts from these cells were assayedfor NF-κB DNA binding-inducing activity by EMSA and only band 2 (B2)corresponding to the 55 kDa protein (FIG. 2C lower panel) was able toelicit NF-κB DNA binding activity while buffer from the beginning or endof the salt gradient failed to activate NF-κB DNA binding activity.

Proteins corresponding to protein bands B1-B6 and blank areas of the gelwere further processed for peptide sequencing. Trypsin digestion of theprotein corresponding to B2 and analysis by electrospray ion trap LC/MSidentified the amino acid sequence of twenty-one peptides. Flagellin(seventy-five percent coverage by the twenty-one peptides) wasunambiguously identified as the protein consistent with inducing NF-κBDNA binding activity (FIG. 3).

EXAMPLE 4 Flagellin is Required to Activate NF-κB in IntestinalEpithelial Cells

To determine if flagellin was indeed the factor that was responsible fortriggering activation of NF-κB after exposure of intestinal epithelialcells to direct bacterial infection or to filtered culture broths ofpathogenic Salmonella, we prepared infectious bacteria and boiled andfiltered culture broths from the non-flagellated E. Coli DH5α,pathogenic S. dublin strain 2229, an isogenic S. dublin 2229 SopE⁻mutant, isogenic S. dublin 2229 SopB⁻ mutant, isogenic S. dublin 2229double SopE⁻/SopB⁻ mutant (strain SE1SB2), S. typhimurium strain 1103,and isogenic S. typhimurium fliC^(::)Tn10 insertion mutant (strain 86)and a S. typhimurium 1103 isogenic double mutant fliC⁻/fljB⁻. SopE is apathogenic Salmonella bacteriophage encoded protein that is injectedinto the host cell and acts as an exchange factor for the small RhoGTPases Rac1 and CdC42 initiating cytoskeleton rearrangements andeventual activation of the MAPK, SAPK and NF-κB pathways (7, 15), whileSopB is a Salmonella protein that functions as an inositol phosphatephosphatase and participates in cytoskeletal rearrangements andstimulates host cell chloride secretion (44). Bacteria and culturebroths were used to challenge HT29 intestinal epithelial cells and WCEextracts were prepared after forty-five minutes and analyzed for NF-κBDNA binding activity by EMSA. Salmonella strains could activate NF-κBwhile Salmonella strains failing to produce flagellin (fliC andfliC⁻/fljB⁻ mutants as indicated) also failed to activate NF-κB (FIGS.4A & B). E. Coli DH5α is non-flagellated and does not produce flagellinfailed to activate NF-κB. We also noticed through numerous experimentsthat S. dublin direct infections always activated NF-κB to a greaterextent than S. typhimurium as observed in FIG. 4A while culture brothsfrom both species activated NF-κB almost equally well (FIG. 4B). Webelieve this difference is due perhaps to S. dublin releasing moreflagellin into the cell culture media than S. typhimurium duringinfection since purification of flagellin from both S. dublin and S.typhimurium and addition of equivalent amounts of chromatographicallypurified flagellin gave similar NF-κB activation profiles. Of note isthe total failure of the double flagellin gene mutants to activate NF-κBas compared to the very minor activation observed in the single Phase Iflagellin fliC::Tn10 insertion mutant (next to last lanes in FIGS. 4A &B) which likely is due to the extremely limited expression of the phaseII flagellin (from fljB), although the strains of Salmonella used heregenetically are unable or rarely shift phases of flagellin production.Since flagellin appears required for activation of the NF-κB pathwayupon direct infection of intestinal epithelial cells it appearedpossible that flagellin may also be the major determinant of other majormitogenic and stress activated signaling pathways activated uponpathogenic Salmonella infection of intestinal epithelial cells. DirectSalmonella infection of intestinal epithelial cells results in JNKactivation (8) and also the activation of NF-κB via IKK (3).

EXAMPLE 5 Flagellin Triggers Activation of the Mitogen Activated ProteinKinase, Stress Activated Protein Kinase and IKK Signaling Pathways

Intestinal epithelial cells act as sentinels for invasion of luminalsurfaces and orchestrate the attraction of effector immune cells to thearea by production of chemokine genes like IL-8 and macrophagechemoattractant protein 1 (MCP1) proinflammatory cytokine genes such asTNFα, IL-1 and IL-6 (1, 4-6). Expression of these genes primarilydepends upon the action of transcription factors that are activated inresponse to the transmission of signals via the MAPK, SAPK and IKKsignaling pathways. Since NF-κB is considered a centralregulator/activator of the proinflammatory gene program we decided toexamine the effect that non-flagellin producing mutant strains ofSalmonella had on activation of the MAPK, SAPK and IKK signalingpathways compared to infection of intestinal epithelial cells withwild-type Salmonella or by exposure of the intestinal epithelial cellsto purified flagellin. Infection of HT29 cells with wild-type S.typhimurium resulted in activation of MAPKs ERK1&2, the SAPKs p38 andJNK and IKK (FIG. 5) as determined by use of activation-indicatingphospho-specific antibodies in immunoblot (IB) analysis orantibody-specific immuno-kinase assays (KA) for JNK and IKK using theirrespective substrates GST-cJun 1-79 and GST-IκBα1-54. Interestingly,MAPK stimulation is transient in nature as activation declines beginningat forty-five minutes while p38, JNK and IKK activity increases withtime through one hour. As seen in FIG. 4, the fliC⁻/fljB⁻ double mutantSalmonella also failed to induce IKK and NF-κB activity (FIG. 5 asindicated). Surprisingly, the fliC⁻/fljB⁻ double mutant Salmonellafailed to induce the SAPKs p38 and JNK and only briefly (fifteenminutes) activated MAPK. This result is puzzling since other Salmonellaproteins such as SopE and SopE2 can activate the small GTPases Rac andCdC42, and these Rho family GTPases have been linked to JNK and p38activation (7, 8, 14, 15) yet appear not to function in the flagellinminus strain.

The fliC⁻/fljB⁻ double mutant Salmonella failed to invade HT29 cellscompared to the wild-type Salmonella strain as determined by gentamycinprotection/invasion assay. The flagellin fliC⁻/fljB⁻ double mutantdisplayed a four orders of magnitude difference in its ability to invadeHT29 cells. To demonstrate this point further, we infected HT29 cellswith either wild-type Salmonella or the fliC⁻/fljB⁻ double mutantSalmonella (strain 134), both strains were transformed with the plasmidpFM10.1 that encodes GFP under the control of the Salmonella ssaHpromoter and only functions once the bacteria has invaded the host cell(10, 36). The wild-type Salmonella clearly was able to infect HT29 cells(GFP, FIG. 5B) while the flagellin mutant bacteria failed to invade HT29cells as evidenced by the lack of GFP expression (FIG. 5B). To determineif flagellin is sufficient or that other bacterially produced proteinsare required for invasion, we added either purified flagellin orsterile-filtered culture broths or a combination of both to HT29 cellsthat were challenged with the Salmonella fliC⁻/fljB⁻ double mutant andassayed for invasion. Intestinal epithelial cells failed to be invadedusing all tested combinations of purified flagellin and/or culturebroths with the fliC⁻/fljB⁻ double mutant strain². There is not believedto be a direct connection between flagellin genes and the effectivenessof the type III secretion system to deliver bacterially producedproteins such as SopE, SopE2 and SipA or other Sip proteins (7, 14, 15,45, 46) that play important roles in initiating bacterialinternalization. Furthermore, to evaluate the effectiveness of flagellinto stimulate p65 (RelA) nuclear localization in intestinal epithelialcells we challenged HT29 cells with purified flagellin and examined p65(RelA) localization using indirect immunofluorescence and found p65(RelA) nuclear localization in nearly every cell (FIG. 5B as indicated).

Purified flagellin (0.5 μg/ml) potently activated NF-κB in HT29 cellssimilar to that observed for TNF (10 ng/ml) treatment of HT29 cells in atime dependent manner (FIG. 6A) when WCE were prepared at the varioustimes as indicated after exposure and assayed for NF-κB DNA bindingactivity in EMSAs. Analysis of the MAPK, SAPK and IKK signaling pathways(FIG. 6B) in these same extracts using activation-specificphospho-antibodies to monitor MAPK and p38 kinase activation orantibody-specific immunoprecipitation kinase assays for JNK and IKKactivities demonstrated that JNK and IKK activity increased through timeto one-hour while p38 and MAPK (ERK1&2) activity peaked at thirtyminutes and began to decline to noticeably lower levels by one-hour(FIG. 6B as indicated). The activation profile of the MAPK, SAPK and IKKsignaling molecules ERK1&2, p38, JNK and IKK in intestinal epithelialcells in response to purified flagellin exposure remarkably resembledthat of intestinal epithelial cells infected with wild-type Salmonella(FIG. 5A). From these observations we conclude that the temporalactivation of the signaling pathways examined here (MAPK, SAPK and IKK),which reflect early events in Salmonella infection, are determinedalmost exclusively by recognition and response of intestinal epithelialcells to flagellin.

We wished to further examine the effect of purified flagellin andflagellin present on Salmonella on the temporal pattern ofproinflammatory cytokine gene expression in intestinal epithelial cellsin order to differentiate the effects of flagellin alone vs. flagellatedSalmonella or non-flagellated Salmonella infection. HT29 cells were leftuntreated, stimulated with TNFα (10 ng/ml), or stimulated with flagellin(0.5 ug/ml), or infected with wild-type Salmonella typhimurium or theSalmonella fliC/fljB double mutant (at MOI of 50). After the indicatedtimes after treatment or infection, HT29 cells were harvested inice-cold PBS and the cell pellets lysed in TRIZOL™ (mono-phasic solutionof phenol and guanidine isothiocyanate) and RNA was purified and used toprepare first-strand cDNA (see Experimental Procedures). Aliquots of thecDNA were used in semi-quantitative RT-PCR reactions using IL1α, IL-1β,IL-8, TNFα, MCP1 and β-actin gene specific primers (sequences availableupon request) and the products were fractionated on ethidium bromidecontaining 1.2% agarose gels. Expression of the known NF-κB target genesIL-1β, IL-8, TNFα and MCP1 was increased in response to TNFα or purifiedflagellin exposure (FIG. 6C). Wild-type Salmonella infection also led toactivation of these same genes although the expression of TNFα and MCP1was transient in comparison and occurred immediately after infection.The Salmonella fliC⁻/fljB⁻ double mutant failed to induce IL-1β, IL-8and TNFα expression, however MCP1 expression was induced, although atlower levels than that induced by wild-type Salmonella, and also, theexpression of MCP1 was not transient in nature but continued throughoutthe time course (9 h) (FIG. 6C). The expression level of β-actin servedas an internal standard for comparison. Interestingly, IL-1α, which isnot an NF-κB target gene was stimulated in response to HT29 cellchallenge by all of the treatments. Obviously, the SalmonellafliC⁻/fljB⁻ double mutant can activate other unknown signaling pathwaysleading to IL-1α expression.

EXAMPLE 6 Flagellin Activates NF-κB DNA Binding in an MyD88-DependentManner

Since flagellin was capable of activating the requisite signalingpathways consistent with proinflammatory gene activation and thisactivity was reminiscent of the action of a cytokine like TNFα thatactivates all cells on which a functional cell surface receptor for itis present (see p65 [RelA] nuclear localization in FIG. 1 and FIG. 5C)we decided to examine the potential of the Toll-like receptors, knownpathogen pattern recognition receptors, to activate the NF-κB pathway inresponse to flagellin exposure. To test this hypothesis we examined theeffect that a dominant-negative MyD88 (aa 152-296) (47) expressingadenovirus had on flagellin-mediated NF-κB activation in HT29 cells.MyD88 is an adapter protein utilized by the IL-1 receptor and all of theknown TLRs, which share homology to IL-1 through their cytoplasmicsignaling domain and is required for immediate activation of the NF-κBpathway (48, 49). Expression of DN-MyD88 in HT29 cells blocked theactivation of NF-κB DNA binding activity assayed by EMSA analysis inresponse to IL-1 or flagellin exposure, consistent with the action of aTLR-mediated activation of NF-κB. To examine this possibility further weinitially used wild-type, MyD88^(−/−) and TLR2^(−/−)TLR4^(−/−) MEFs (agift of S. Akira, Univ. of Osaka, JA) to verify the role of MyD88 and toexamine the potential role of two of the TLRs to respond to flagellin orto direct wild-type Salmonella infection and lead to NF-κB activation(FIG. 7). Wild-type Salmonella infection activates NF-κB potently inboth the wild-type and TLR deficient MEFs (lanes 2 & 15) but thisactivation is somewhat defective in the MyD88 deficient MEFs (lane 10).Challenge of all three types of cells with concentrated sterile-filteredwild-type S. dublin or the double SopE⁻/SopB⁻ isogenic mutant S. dublinstrain SE1SB2 culture broths activated NF-κB in wild-type MEFs andTLR2/4 double deficient cells but failed to activate NF-κB in MyD88deficient cells (compare lanes 11 and 12 with lanes 3, 4, 6, 7, 16 and17). NF-κB was potently activated in wild-type MEFs by exposure topurified flagellin (0.5 μg/ml) and therefore eliminated the possibilitythat LPS played a role in NF-κB activation in these experiments. Theexclusion of LPS as a major contributor to NF-κB activation is alsoprovided by the potent activation of the TLR2/4 double deficient MEFs(lanes 16 & 17). TLRs 2 and 4 respond to bacterial lipopeptides,peptidoglycans, certain LPSs and gram negative LPS respectively (50-52).IL-1 stimulation verified the functional requirement of MyD88 intransmission of IL-1 and flagellin-mediated signals.

To further define a possible role for the TLRs in flagellin recognitionwe assayed for the ability of overexpressed TLRs to activate NF-κB incells that normally respond poorly to flagellin exposure. Choosing cellsthat responded slightly to purified flagellin ensured that the signalingcomponents and adapters that flagellin uses were present and functionaland that the limiting factor was likely only to be the receptor thatresponds to flagellin. We found that HeLa cells and HEK293T cellsactivated NF-κB DNA binding activity in response to IL-1 stimulation butpoorly to flagellin exposure and we chose HEK293T cells to use furtherbecause of their greater transfection efficiency. Amino-terminus FLAGepitope-tagged TLRs 1-9 (kind gifts of R. Medzhitov, Yale Univ. and R.Ulevitch, TSR1) (42, 43) were overexpressed in HEK 293T cells intransient transfections along with the 2×-NF-κB-dependent promoterdriven luciferase reporter gene and the expression of luciferase inresponse to no treatment, flagellin (0.5 μg/ml) or TNFα (10 ng/ml) wasdetermined. TLR5 was the only TLR whose expression resulted in anoticeable response to flagellin challenge of the cells (Table 1).

To further determine the likelihood of TLR5 being the TLR through whichflagellin activated NF-κB, we constructed dominant-negative signalingmutations by deletion of the carboxyl portion of each TLR to a conservedtryptophan in the TIR domain. A similar mutation in the IL-1 receptorabrogates its ability to lead to NF-κB activation (54, 55). Each DN-TLRalong with a reverse cloned TLR5 (AS-TLR5) was cloned into the mammalianexpression vector pcDNA3.1 (Invitrogen). All mutant proteins wereexpressed well. Each DN-TLR mammalian expression vector and emptyexpression vector along with 2× NF-κB Luc was transfected as previouslydescribed (3) into HT29 cells which respond very well to flagellin. Thetransfected cells were left untreated, stimulated with TNFα (10 ng/ml)or with purified flagellin (0.5 g/ml). Reporter gene expression wasobserved not to be affected by DN-TLR expression in response to TNFαstimulation of transfected cells (FIG. 8A); however, only expression ofeither the DN-TLR5 or an antisense TLR5 construct resulted in a nearlyfifty percent and twenty-five percent inhibition of flagellin-mediatedreporter gene activation respectively (FIG. 8B), while DN-TLR2 also wasfound to mildly inhibit flagellin-mediated reporter expression. Theseresults imply that TLR5 takes part in cell surface recognition offlagellin and initiates the signaling pathway leading to NF-κBactivation. The effect of DN-TLR2 on NF-κB-dependent reporter geneactivation may be non-specific since its expression also inhibitedTNFα-mediated reporter activation as compared to the other DN-TLRs.DN-TLR2 may also compete for an unknown adapter protein that both TLR2and TLR5 might share. In any event, TLR2 and TLR4 were shown by theresults presented in FIG. 7 not to be required for flagellin-mediatedactivation of NF-κB.

EXAMPLE 7 Flagellin-Mediated Activation of NF-κB Leads to IncreasedExpression of a Subset of TLRs

Stimulation of intestinal epithelial cells with S. typhimurium or withpurified flagellin led to activation of the proinflammatory gene program(FIG. 6C). We wished to examine whether or not expression of TLR geneswould also be altered in flagellin-stimulated cells. HT29 cells weretreated with purified flagellin (0.5 μg/ml) and total RNA was isolatedfrom non-treated and treated cells three hours after stimulation andused to make first-strand cDNA. Semi-quantitative RT-PCR usinggene-specific primers for each of the TLRs and first-strand cDNAprepared from non-stimulated or flagellin stimulated cells was used togenerate DNA products that were fractionated on ethidium bromidecontaining 1.2% agarose gels. TLRs 2, 3 and 7 were increased inexpression after flagellin stimulation (FIG. 9). The expression patternof the other TLRs remained unchanged, β-actin expression served as aninternal abundance control.

TLR5 is expressed in cells that don't respond well to flagellin. Thisstudy and others (22, 33) have identified TLR5 as the likely TLR throughwhich flagellin activates NF-κB. Previous reports made no determinationon the presence or abundance of TLR5 in the cells that they used toascertain its function (22, 33). We wished to determine if TLR5 proteinabundance was absent or greatly decreased in cells that failed torespond or responded poorly to challenge by flagellin. TLR5 abundance ina number of cell lines was examined by immunoblot analysis using aTLR5-specific antibody and compared with the ability of purifiedflagellin to induce NF-κB DNA binding activity of WCEs prepared fromthem. Intestinal epithelial cell lines T84 and HT29 were used as was thelung adenocarcinoma cell line A549, the human cervical adenocarcinomacell line HeLa, the human embryonic kidney cell line expressing large Tantigen HEK293T, and the glioblastoma cell line T98G. TLR5 protein wasdetected in all cell lines examined by immunoblot with TLR5-specificantibody (FIG. 10A). T84 cells exhibited the highest abundance whileexpression levels of the other cell lines were similar and appeared notto differ by more than two-fold (FIG. 10A). NF-κB DNA binding activityin non-stimulated, TNFα and flagellin stimulated cells was analyzed byEMSA assays of WCEs prepared from each cell type (FIG. 10B). HT29 andA549 cells responded strongly to flagellin and to TNFα stimulation whileHeLa, 293T and T98G cells responded poorly (HeLa, 293T) or not at all(T98G) to flagellin stimulation. The authenticity of the NF-κB DNAbinding complex was determined using p65-specific antibody to supershiftthe NF-κB DNA:protein complex. It is of interest that some cells thatexpress TLR5 either do not respond at all or do so very poorly. This maybe due to either lack of receptor presence at the plasma membrane andintracellular localization, inactivating or detrimental mutations in theTLR5 gene in these cell lines or lack of or low abundance of a requiredco-receptor or adapter protein (as is the case in some cells for TLR4and its co-receptor/adapter MD2 (30, 56, 57)). IL-1 can activate NF-κBDNA binding activity in all of the examined cell lines so it appearsthat the signaling apparatus downstream of MyD88 to NF-κB is intact.

EXAMPLE 8 Isolation of Recombinant Flagellin

In order to confirm that recombinant flagellin was able to induce NF-κB,it was tested for activity using reporter cells carryingNF-κB-responsive luciferase (luc). The reporter construct contains threeNF-κB-binding sites from the E-selectin promoter combined with the Hsp70minimal promoter and is routinely used for the detection of NF-κB.Luciferase activity was measured in cell lysates 6 hours after additionof flagellin into the medium. TNFα was used as a positive control. Theresults of a representative experiment are shown in FIG. 13 and indicatethat recombinant flagellin is capable of NF-κB activation.

EXAMPLE 9 Flagellin Delays Mouse Death Caused by IR-Induced GI Syndrome

As indicated above, flagellin is a potent activator of NF-κB andpresumably can act as an inhibitor of apoptotic death. Since cytokinescapable of inducing NF-κB act as radioprotectants, we tested whetherflagellin might also serve as a radioprotectant.

Whole body irradiation of mice with 15 Gy gamma radiation leads to deathwithin 8 days from GI syndrome providing a conventional model ofradiation induced damage of GI tract (see above). To test whetherflagellin is capable of protecting GI epithelium from IR, we tested theeffect of i.v.-injected flagellin on the dynamics of mouse lethalityafter 15 Gy of radiation. We used a range of flagellin doses, all ofwhich were significantly lower than the highest tolerable dose knownfrom literature (300 μg/mouse, Eaves-Pyles T, et al 2001b). Irradiationwas done 4 hours post treatment. The results of a representativeexperiment are shown in FIG. 14. As expected, control irradiated mice(that received PBS i.v.) died between 5 and 8 days post-treatment, whileanimals that received flagellin lived significantly longer; theextension of animal survival correlated with the dose of flagellin.Pathomorphological analysis of the small intestine on day 7 afterirradiation reveals dramatic difference between flagellin-treated andcontrol groups (FIG. 15). Intravenous, intraperitoneal and subcutaneousdelivery of 0.2 mg/kg of flagellin followed by 13 Gy irradiationafforded similar degree of protection, leading to 85-90% 30-day survivalof mice (data not shown). Experiments were performed essentially asdescribed above for optimal dosage experiments but with 13 Gyirradiation and varied routes of delivery.

EXAMPLE 10 Flagellin Rescues Mice from Lethal IR-Induced HematopoieticSyndrome

We next tested whether flagellin has an effect on mouse IR-induced deathfrom HP syndrome that is experimentally induced by lower radiation doses(usually up to 11 Gy) that are incapable of causing lethal GI toxicity.The experiments were done similarly to the above-described ones (FIGS.14 and 15), however, instead of 15 Gy, mice received 10 Gy, the dosethat caused 100% killing in control group by day 13 (FIG. 16).Flagellin-treated group (5 μg/mouse) showed complete protection fromthis dose of IR indicating that flagellin-mediated radioprotection actsnot only against GI but also against HP IR-induced syndromes.

EXAMPLE 11 Time Dependence on the Protective Effect of Flagellin

In order to estimate the dependence of radioprotective activity offlagellin on the time of treatment by injecting mice at different timesbefore 13 Gy of gamma irradiation. The results of one of suchexperiments is shown in FIG. 17. The obtained results show thatflagellin is effective as radioprotectant from 13 Gy if injected 1-4 hbefore treatment but is no longer effective if injected 24 h beforeirradiation.

In order to estimate the dependence of radioprotective activity offlagellin on the time of treatment, mice were injected at several timepoints relative to the moment of gamma-irradiation. Experiments weredone essentially as explained above, using intraperitoneal injection of5 μg/mouse (0.2 mg/kg) of CBLB-501 or, for control mice, 5 μg/mouse (0.2mg/kg) of bacterial RNA polymerase. The experiments were performed onNIH-Swiss mouse strain. The results show that flagellin −501 provides˜90% survival after 13 Gy irradiation if injected at 1 or 2 hours beforetreatment (FIG. 17). Only −1 h graph is shown for clarity, however, bothtimepoints (−1 and −2 h) provide similar degree and dynamics ofsurvival. 4 h timepoint shows somewhat lower protection. Flagellininjected 24 hours before irradiation had no protective effect against 13Gy induced death.

Interestingly, administration of flagellin 24 hours before 10 Gygamma-irradiation provided 100% protection. While 13 Gy irradiation inmice primarily induces death from GI syndrome, 10 Gy-induced death ismostly mediated by hematopoietic syndrome. Accordingly, such long-termprotection from 10 Gy irradiation may be mediated by enhancedproliferation or survival of hematopoietic stem cell induced byflagellin and/or long-living secondary cytokines.

EXAMPLE 12 Determination of LD_(50/30), LD_(50/7) and DMF for Flagellin

We obtained an estimate of radiation dose-dependent protection forflagellin. As shown above (FIG. 17), treatment with flagellin wassufficient for 100% protection against 10 Gy gamma-irradiation (thisdose causes death from hematopoietic syndrome) and 90% 30-day survivalat 13 Gy (both hematopoietic and GI syndromes). Experiments wereperformed as described above, using flagellin 5 μg/mouse (0.2 mg/kg),intraperitoneally injected 1 h before irradiation.

At 15 Gy, however, 100% 7-day survival was followed by delayed deathafter 13 days (0% 30-day survival), while control group had fullysuccumbed to GI syndrome by day 7 (FIG. 18). The kinetics of CBLB-501treated group mortality after 15 Gy irradiation is reminiscent of suchof control group at 10 Gy, hinting at death caused by hematopoieticsyndrome. The results provide an estimate of flagellin LD_(50/30) around13.5-14 Gy and DMF₃₀ of about 1.75-1.8. This degree of radioprotectionis significantly higher than any reported for a natural compound.

1. A method of protecting a mammal from ionizing radiation sufficient tocause gastrointestinal syndrome or hematopoietic syndrome comprisingadministering to a mammal in need thereof a composition comprisingrecombinant flagellin.
 2. The method of claim 1, wherein the compositionis administered in combination with a radioprotectant.
 3. The method ofclaim 2, wherein the radioprotectant is an antioxidant.
 4. The method ofclaim 3, wherein the antioxidant is selected from the group consistingof amifostine and vitamin E.
 5. The method of claim 2, wherein theradioprotectant is a cytokine.
 6. The method of claim 5, wherein thecytokine is stem cell factor.
 7. The method of claim 1, wherein thecomposition is administered prior to, together with, or after radiation.8. The method of claim 1, wherein the composition is administered incombination with a growth factor.
 9. The method of claim 8, wherein thegrowth factor is keratinocyte growth factor.
 10. The method of claim 1,wherein the composition is administered in combination with a steroid.11. The method of claim 10, wherein the steroid is 5-androstenediol. 12.The method of claim 1, wherein the composition is administered incombination with ammonium trichloro(dioxoethylene-O,O′)tellurate. 13.The method as in one of claims 1, 2-6, 7, or 8-12, wherein the mammal isa human.